Amino-substituted hypocrellins for use as sonosensitizers

ABSTRACT

The invention is novel amino-substituted hypocrellins, such as demethoxylated hypocrellin B, that are sonosensitizers, and their therapeutic use.

TECHNICAL FIELD OF THE INVENTION

The invention involves compositions and methods for treating diseasesand the like by administering compounds that are sonosensitizers. Thecompositions comprise amino-substituted hypocrellins, preferablydemethoxylated hypocrellin B.

BACKGROUND OF THE INVENTION

Treatment for cancer has traditionally encompassed three mainstrategies: surgery, chemotherapy, and radiotherapy. Althoughconsiderable progress in these areas has been attained, the search formore effective and safe alternative treatments continues. In thisregard, photodynamic therapy (PDT) and sonodynamic therapy (SDT)represent a promising new approach(es) for the treatment of cancer.These therapies involve systemic or topical administration of asensitizer, followed by its activation by light of a specific wavelength(PDT), or activation by sound of a specific frequency (SDT). Theactivation of the sensitizer leads to the production of activated oxygenand radical species that initiate a cascade of biochemical reactions,resulting in direct cell destruction, damage to the tumor vasculatureand immune inflammatory responses. Lipson, et al. were the first to usephotodynamic therapy, in 1966 at the Mayo Clinic [Proc. IX Internat.Cancer Congress, page 393 (1966)].

The photosensitizing and therapeutic properties of perylenequinonoidpigments (PQPs), such as hypocrellins, in biological systems have beenrecognized during the past two decades. See Diwu, et al., J. Photochem.Photobiol. A: Chem., 64:273 (1992); Zhang et al., (1989); and Wan, etal., “Hypocrellin A, a new drug for photochemotherapy,” Kexue Tongbao(English edition) 26:1040 (1981). Because of the difficulty ofcollecting sufficient activated photosensitizer at the site of action,none of the previously known photosensitizers have gained widespread useas therapeutics.

The importance of sonodynamic therapy (SDT) lies ultimately in itssimilarity to PDT, an elegant and effective tumor treatment whosesuccess is due to the use of light and drug in combination, i.e., twotreatment elements, neither of which has toxic effects by itself(Marcus, 1992). The principal impetus for the development of SDT hasbeen improvement upon PDT's dosimetric shortcomings. PDT is currentlyrestricted to use with superficial tumors. Its use on tumors deep withinthe body requires interstitial irradiation that increases the complexityof the treatment and compromises its noninvasive nature. SDT provides ameans to reach such tumors, since ultrasound propagates easily throughseveral centimeters of tissue, and like light, can be focusedprincipally on the tumor mass where it activates the sonosensitizingcompound. Targeted SDT offers the possibility of improving the toleranceof this therapy by further restricting its effects to the target tissue.

While these discoveries represent significant advances, two seriousdeficiencies remain in the development of experimental SDT. Asubstantial problem is the lack of sonodynamic agents with favorableclinical properties. Porphyrins are known to cause significant cutaneousphotosensitivity (Estey et al., 1996), doxorubicin is cardiotoxic (Myerset al., 1976), and DMSO, DMF and MMF are hepatotoxic (Misik and Riesz,1996). New sensitizers with better sonodynamic properties, which havemilder side effects and which are rapidly cleared, would greatly improvethe clinical application of SDT. A further problem is the lack ofstandardization in the conditions used for evaluating sonodynamicagents.

Potential sonodynamic agents have been tested following exposure toultrasound intensities ranging from 0.25 W/cm² to 40 W/cm², andfrequencies from 500 MHz to 1 MHz (Harrison et al., 1991; Sasaki et al.,1998). Though in vivo use would seem to require greater energies due toroughly isotropic dissipation of the ultrasonic energy, little efforthas been made to compare experimental conditions in vitro with those invivo. Where one group will find evidence of sonodynamic effect,different investigators do not under apparently similar conditions.Development of standard insonation and assay systems compatible withclinical use will permit a more rigorous assessment of the sonodynamiceffects of current and future sonosensitizers.

Sonodynamic activation of sensitizers has been found to be useful sinceultrasound has the appropriate tissue attenuation coefficient forpenetrating intervening tissues to reach desired treatment volumes,while retaining the ability to focus energy on reasonably small volumes.Diagnostic ultrasound is a well accepted, non-invasive procedure widelyused in the developed world, and is considered safe even for fetalimaging. The frequency range of diagnostic ultrasound lies between 100kHz-12 MHz, while 50 kHz sound provides enough energy to effect cellulardestruction through microregional cavitation.

Sonodynamic therapy provides treatment strategies unavailable instandard photodynamic therapy, due to the limited tissue penetration ofvisible light. One example would be the treatment of newly diagnosedbreast cancer, where local and regional spread of micrometastaticdisease remains clinically undetectable. Using immunoconjugates(anti-breast cancer Mab-sonosensitizer hybrids), it would betheoretically possible to selectively eradicate micrometastases in theabsence of normal tissue damage.

Beyond these basic properties shared with other waves, ultrasoundexhibits unique properties when propagating through water. Above acertain threshold intensity, propagation of ultrasound waves throughwater elicits an effect termed ‘cavitation’ (Rayleigh, 1917; Connollyand Fox, 1954). Cavitation involves the formation of small bubbles or‘cavities’ in the water during the rarefaction half of the wave cycle,followed by the collapse of these bubbles during the compression half ofthe cycle (Putterman, 1995). Cavities focus the energy of the incidentultrasonic radiation by many orders of magnitude (Hiller et al., 1992).The consequence is that regions of cavitation in water are sites ofextremely high temperature and pressure. Estimates of the temperaturesgenerated in a collapsing cavity range from 5000K to 10⁶K (Suslick etal. 1986; Flint and Suslick, 1991; Misik and Riesz, 1995; Kaiser, 1995).

The biological effects of exposure to ultrasound are the result of itsphysical and chemical effects. The most obvious biological effects ofultrasound treatment stem from heating of the medium through which itpasses. Such heating is exploited during physiotherapy to help healinjured tissues. (Lehmann et al., 1967; Patrick, 1966), and has beeninvestigated as a possible modality for tumor treatment. This is due tothe sensitivity of many tumours to hyperthermia, a state in which tissuetemperatures are elevated above 42° C. (Doss and McCabe, 1976; Marmor etal., 1979; Sculier and Klastersky, 1981; Bleehen, 1982; Hynynen andLulu, 1990). Ultrasound has also been used in combination with radiationtherapy to improve treatment response in vivo compared to radiotherapyalone (Clarke et al., 1970; Repacholi et al., 1971; Mitsumori et al.,1996). A principal danger in the use of ultrasound for therapeuticpurposes is the formation of ‘hotspots’ due to regions of constructiveinterference and preferential absorption of ultrasonic energy by boneregions with low curvature radii⁺ (Lehmann et al., 1967; Linke et al.,1973). These hotspots can cause serious damage to nearby tissues (Hill,1968; Bruno et al., 1998).

As is the case of hematoporphyrin derivatives, natural PQPs do notthemselves exhibit absorptivity longer than 600 nm, a characteristicthat inherently predicts a decreased capability of activation as tissuedepth increases beyond 3-5 mm. This means that the natural PQPs are notsufficiently strong for photodynamic therapy, and this limits theirphotodynamic therapy applications.

Deficiencies of current porphyrin and PQP photosensitizers forphotodynamic therapy have stimulated the development of a series ofsecond generation compounds which have improved properties with respectto light absorption in the red spectral range, purity, pharmacokinetics,and reduced cutaneous photosensitivity. These deficiencies also lead toinvestigating other forms of activating the sensitizer, e.g., activationusing sound waves.

It has been noted that some photosensitizing compounds also confercytotoxicity when they are activated by ultrasound. Sonodynamicactivation of photosensitizers (as used hereinafter, “sonosensitizers” )is clinically relevant, since ultrasound has the appropriate tissueattenuation coefficient for penetrating intervening tissues to reachdesired treatment volumes, while retaining the ability to focus energyon reasonably small volumes. Diagnostic ultrasound is a well accepted,non-invasive procedure widely used in the developed world, and isconsidered safe, even for fetal imaging. The frequency range ofdiagnostic ultrasound lies between 100 kHz-12 MHz, while 50 kHz soundwill provide enough energy to effect cellular destruction throughmicroregional cavitation. The molecular mechanisms of sonodynamiccytotoxicity are currently not well understood, however a role forsinglet oxygen has been postulated. Sonodynamic effects of porphyrinsand porphyrin analogs in cell culture and experimental animal tumorshave been reported. Sonodynamic therapy provides treatment strategiesunavailable in standard photodynamic therapy, due to the limited tissuepenetration of red, visible light. The sensitizers under investigationinclude proprietary molecules for which this use has never beenreported. Proof-of-concept has been shown for 7 perylenequinones, usedto sonosensitize human promyelocytic leukemia (HL-60) cells in vitro.

The ultrasound generator is clinically licensed in Europe and NorthAmerica for physiotherapy use. It is readily adapted to in vitrostudies, and is used in its “off-the-shelf” configuration in cancertherapy. A wide variety of neoplastic diseases, as well as infectiousdiseases, autoimmune and hyperproliferative diseases, or any diseasewhere targeted therapy is appropriate, could benefit from thesonodynamic approach.

SUMMARY OF THE INVENTION

In accordance with the present invention, hypocrellin derivatives, suchas amino-substituted hypocrellins, including but not limited todemethoxylated hypocrellin B (DMHB), have sonosensitizing properties andmay be used to treat diseases and other conditions. Moreover, the HBderivatives of the present invention may be conjugated to a deliverymoiety to enhance the ability of the PQP derivative to target predetermined cells or structures in vitro or in vivo.

The methods and compositions of the present invention, activated bysound, exhibit substantial absorption in the therapeutic frequencies ofultrasound; produce high singlet oxygen yield; can be produced in pure,monomeric form; may be derivatized to optimize properties of ultrasoundactivation, tissue biodistribution, and toxicity; and are rapidlyexcreted. They afford nuclear targeting by covalent attachment to DNAminor-groove binding agents, such as stapled lexotropins, to enhancesonotoxicity. They are not genotoxic. This trait is important in thecontext of treatment-related secondary malignancies.

Many PQP properties are summarized in Diwu, et al., J. Photochem.Photobiol. A: Chem., 64:273 (1992). Some perylenequinones are alsopotent inhibitors of certain viruses, particularly humanimmunodeficiency virus (HIV), and also the enzyme protein kinase C(PKC). Both anti-HIV and anti-PKC activities of certain PQPs arelight-dependent, a phenomenon implicated in the photodynamic therapy ofcancers [Diwu, et al., Biochem. Pharmacol., 47:373-389 (1994)]. The Diwuet al paper also discloses the successful conjugation of HB to aprotein.

The sonosensitizing compounds of the present invention, whenadministered systemically, distribute throughout the body. Over a shortperiod, ranging from hours to days, the compounds clear from normaltissues, but are selectively retained by rapidly proliferating cells(e.g., cancer cells or psoriasis lesions) for up to several days. Theamino-substituted hypocrellins (e.g., DMHBs) of the present inventionare inactive and non-toxic until activated, e.g., exposed to light in aspecific wavelength range or to sound in a specific frequency range.

The compounds of the present invention are also beneficialtherapeutically due to their dual selectivity. The compounds of thepresent invention are selective in their ability to preferentiallylocalize the drug at the site of a predetermined target, such as acancer cell, and they are selective in that precise delivery of lightand/or sound can be confined to a specific area.

The methods and compositions of the present invention, when administeredin vivo, such as intravenously, distribute throughout the body. Insubsequent hours, and sometimes days, the compositions containing atleast one perylenequinone derivative begin to clear from normal tissues,but are selectively retained for up to several days byhyperproliferating cells, such as cancer cells. The HB remains inactiveand non-toxic until it is activated. In accordance with the presentinvention, the HB may be activated by sound. The hyperproliferatingcells, now containing or contacted with a HB compound, may be exposed toan activation source, e.g., sound of an appropriate frequency. Exposingthe site containing the hyperproliferating cells with the activationsource permits selective activation of the retained perylenequinonederivative, which in turn initiates local necrosis or apoptosis in thehyperproliferating cell tissue leading to cell death.

In combination with the delivery system according to the presentinvention, the compositions and methods of the present invention permitincreased selectivity by preferential localization of theperylenequinone derivative at the site of the targeted cells, and permitincreased selectivity by confining the activation source to a specificarea, e.g., sound confined to a discrete area.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows several structures for the demethoxylated HB compounds ofthe present invention, where R₁, R₂, R₃, R₄ are OCH₃ or NHCH₂Ar (Ar arephenyl or pyridyl group), NHCH(CH₂)_(n) (where —CH(CH₂)_(n) arealicyclic group and n=3,4,5,6). 2-BA-2-DMHB is where R₁, R₂, R₃are OCH₃,and R₄ is NH(CH₂)₃CH₃. Alternatively, R₁, R₂, R₃, R₄ may be OCH₃orNHCH₂(CH₂)_(n)Ar, wherein Ar is a phenyl, naphthyl, polycyclic aromaticor a heterocyclic moiety, and n is 0-12.

FIG. 2 shows that tumor growth is inhibited by SDT with DMHB.

MODES FOR CARRYING OUT THE INVENTION

The present invention comprises the use of amino-substitutedhypocrellins, e.g., the demethoxylated hypocrellin compounds of FIG. 1,as sonodynamic agents, and the use of these compounds according to theinvention as therapeutics.

The present invention includes a composition and method for treating apre-determined disease or condition comprising administering atherapeutic amount of a composition comprising a compound of thestructure shown in FIG. 1, allowing the compound to distribute to aportion of the body, preferably throughout the body, and activating thecompound in an area containing hyperproliferating cells. In preferredembodiments of the invention, the activating step includes activatingthe DMHB compound with sound.

The present invention also includes methods and compositions thatinvolve the topical application of a composition according to theinvention, and activating the active agent in the composition. Inpreferred embodiments of the invention, the active agent is suitable fortreating dermatological conditions, including but not limited to acneand hair removal.

The present invention also includes methods and compositions thatinvolve the use of a composition according to the invention as ananti-bacterial agent in dental applications. In these embodiments of theinvention, the active agent is formulated into a liquid composition,such as a mouthwash, contacting a tooth or teeth with the composition,and activating the active agent in the composition. In this embodimentof the invention, the composition is useful in treating cariotosis andthe like. In preferred embodiments of the invention, the conjugate maybe activated by sonoactivation.

The invention also comprises a method of treating a disease byadministering a therapeutically sufficient amount of at least one DMHBaccording to the invention, and activating the derivative(s) usingsonoactivation. Typically, the DMHB compound may be activated byexposing the compound to a pre-determined sound frequency.

The invention also includes sonosensitive compounds that furthercomprise a cleavable linker, said linker being cleavable in vivo. Inaccordance with the present invention, the cleavable linker may bechosen to alter one or more properties of the compound, including butnot limited to solubility, stability, absorption, and the like.Cleavable linkers include, but are not limited to, polyamides andsugars.

In a preferred embodiment of the invention, the HB is anamino-sustituted derivative of hypocrellin B. In the most preferredembodiments of the invention, the HB derivatives are demethoxylatedhypocrellins (see FIG. 1), where R₁, R₂, R₃, R₄ are OCH₃ or NHCH₂Ar (Arare phenyl or pyridyl group), NHCH(CH₂)_(n) (where —CH(CH₂)_(n) arealicyclic group and n=3,4,5,6). 2-BA-2-DMHB is where R₁, R₂, R₃ areOCH₃, and R₄ is NH(CH₂)₃CH₃. Alternatively, R₁, R₂, R₃, R₄ may be OCH₃or NHCH₂(CH₂)_(n)Ar, wherein Ar is a phenyl, naphthyl, polycyclicaromatic or a heterocyclic moiety, and n is 0-12.

The compounds of the present invention may be produced by any methodthat results in a purified or substantially purified compound, or in acompound that is useful as a photodynamic or sonodynamic agent. Thecompounds of the present invention may also form a compositioncomprising a cocktail of compounds, e.g., more than one compound. Thesemethods are well known in the art, e.g., Liu, et al., “Synthetic studiesin novel hypocrellin B derivatives,” Tetrahedron, 49:10785 (1993); andDiwu, et al., Anti-Cancer Drug Design, 8:129-143 (1993).

The amino-substituted HB compounds of the present invention areparticularly suited for therapeutic use because they exhibit excellentsonodynamic activity in a frequency range from about 1 MHz to about 3MHz; are low molecular weight, typically from about 550 daltons to about880 daltons); are available in pure monomeric form; exhibit rapid serumand skin clearance; have negligible cytotoxicity in vitro and in vivo;have excellent sonopotentiation (e.g., two orders of magnitude), so thesafety margin in use is excellent; potent inhibitors of protein kinases;confer apoptotic cell death in vitro and in vivo; exhibit nogenotoxicity; exhibit excellent tumor control; may be molecularlyconfigured for targeted delivery; may be targeted to nuclear regions tofurther augment sonotoxicity; and the parent hypocrellins are amenableto site-specific modification, so that many derivatives may be formed,derivatives with varying degrees of sonosensitizing characteristics.

As used herein, “disease” refers to the management, diagnosis, and/orpalliation of any mammalian (including human) disease, disorder, malady,or condition that can be treated by photodynamic therapy. “Disease”includes but is not limited to cancer and its metastases, such as skincancer; growths or tumors, and their metastases; tumors and tumor cells,such as sarcomas and carcinomas, including solid tumors, blood-bornetumors, and tumors found in nasal passages, the bladder, the esophagus,or lung, including the bronchi; viruses, including retroviruses;bacterial diseases; fungal diseases; and dermatological conditions ordisorders, such as lesions of the vulva, keloid, vitiligo, psoriasis,benign tumors, endometriosis, Barett's esophagus, Tinea capitis, andlichen amyloidosis.

As used herein, “administering” and “delivering” refers to any actionthat results in exposing or contacting one or more DMHB derivatives witha predetermined cell, cells, or tissue, typically mammalian. As usedherein, administering or delivering may be conducted in vivo, in vitro,or ex vivo. For example, a composition may be administered by injectionor through an endoscope. Administering also includes the directapplication to cells of a composition according to the presentinvention. For example, during the course of surgery, tumor cells may beexposed. In accordance with an embodiment of the invention, theseexposed cells (or tumors) may be exposed directly to a composition ofthe present invention, e.g., by washing or irrigating the surgical siteand/or the cells.

As used herein, activation, activating, or similar terms refers to theuse of sound frequency to make a compound or portion of a compound morereactive. Any method for applying a sound source to a perylenequinonederivative may be used in accordance with the present invention, e.g.,direct application, an ultrasound machine, focused ultrasound,high-intensity focused ultrasound, to name a few.

Upon application of the appropriate sound, the sensitizers canchemically (e.g., through oxidation, reduction and the like) change intoa form that is toxic to the surrounding tissue. For example, followingexcitation of a sonosensitizer to a long-lived excited triplet state, atargeted tumor is destroyed either by the highly reactive singlet oxygenspecies (a Type II mechanism) and/or by free radical products (a Type Imechanism) generated by quantum energy transfer. Major biological targetmolecules of the singlet oxygen species and/or free radical productsinclude nucleic acids, enzymes and cell membranes. A secondarytherapeutic effect of the present methods involves the release ofpathophysiologic products, such as prostaglandins, thromboxanes andleukotrienes, by tissue exposed to the effects of activatedphotosensitizers. Thus, it will be apparent to one skilled in the artthat careful targeting of the sonoactive agents is of paramountimportance to achieve therapeutic effects without eliciting toxemias.

As used herein, “sonosensitizing potential” refers to the property ofthe compound(s) to exert sound-mediated toxicity in excess of its(their) inherent unactivated toxicity. In a preferred embodiment of theinvention, the activation factor may be calculated as the ratio of theLD₅₀ of cells treated without activation to the LD50 of the cellstreated with an activated compound (drug LD₅₀ divided by activated drugLD₅₀). Where the term “LD₅₀” has been used above, the term “IC₅₀” may besubstituted, to address the bioassays that concern metabolic activityrather than the endpoint of lethality, loss of reproductive capability,or clonogenic death.

In accordance with the present invention, a desirable compound of thepresent invention is one that is non-toxic (or of low toxicity) at highdrug concentrations without activation, i.e., without sound, and istoxic at low concentrations when sound of the appropriate frequency, isapplied. As is recognized by those skilled in the art, the mostdesirable compounds are those that provide a wide range of non-toxicdoses in an unactivated state, as this characteristic provides anincreased safety factor for the patient.

As used herein, physiologically acceptable fluid refers to any fluid oradditive suitable for combination with a composition containing a PQPderivative. Typically these fluids are used as a diluent or carrier.Exemplary physiologically acceptable fluids include but are not limitedto preservative solutions, saline solution, an isotonic (about 0.9%)saline solution, or about a 5% albumin solution or suspension. It isintended that the present invention is not to be limited by the type ofphysiologically acceptable fluid used. The composition may also includepharmaceutically acceptable carriers. Pharmaceutically accepted carriersinclude but are not limited to saline, sterile water, phosphate bufferedsaline, and the like. Other buffering agents, dispersing agents, andinert non-toxic substances suitable for delivery to a patient may beincluded in the compositions of the present invention. The compositionsmay be solutions, suspensions or any appropriate formulation suitablefor administration, and are typically sterile and free of undesirableparticulate matter. The compositions may be sterilized by conventionalsterilization techniques.

In accordance with a method of the invention, the composition containinga sonosensitizer of the present invention may be administered to thepatient by any biologically suitable route. For example, thesonosensitizer may be introduced into the patient by intravenous,subcutaneous, intraperitoneal, intrathecal, intravesical, intradermal,intramuscular, or intralymphatic routes. The composition may be insolution, tablet, aerosol, or multi-phase formulation forms. Liposomes,long-circulating liposomes, immunoliposomes, biodegradable microspheres,micelles, or the like may also be used as a carrier, vehicle, ordelivery system. Furthermore, using ex vivo procedures well known in theart, blood or serum from the patient may be removed from the patient;optionally, it may be desirable to purify the antigen in the patient'sblood; the blood or serum may then be mixed with a composition thatincludes a sonosensitizer according to the invention; and the treatedblood or serum is returned to the patient. The invention should not belimited to any particular method of introducing the sonosensitizer intothe patient.

The amino-substituted HB derivatives of the present invention may alsobe used in conjunction with and conjugated to a number of othercompounds, signaling agents, enhancers, and/or targeting agents. Forexample, a hypocrellin derivative of the present invention may beconjugated to an antibody, preferably a monoclonal antibody. Inaccordance with the present invention, the binding agent includes anyDNA minor-groove targeting agent, such as lexotropsin or netropsin,preferably to enhance the toxicity through targeting the cell nucleus.Suitable enhancers include but are not limited to pKa modifiers, hypoxiccell radiosensitizers, and bioreductively activated anti-neoplasticagents, such as mitomycin C (preferably to effect or potentiate thetoxicity of the compound in hypoxic cells or microorganisms). Suitablesignaling agents include but are not limited to markers of apoptoticcell death or necrotic cell death, or regulatory molecules endogenous tocell cycle control or delay, preferably to potentiate the phototoxicityor sonotoxicity of the compound(s) by induction of apoptotic or necroticcell death, or by inhibition of the repair of any form of lethal orpotentially lethal damage (PLD).

In accordance with the present invention, the PQP derivatives may befunctionalized, e.g., include reactive groups including but not limitedto an acid, hydroxyl, an acid halide (preferably bromide), a hydrazine,a thiol, or a primary amine. The binding reagent may include reactivegroups including but not limited to amino acids, such as cysteine,lysine, aspartic acid, glutamic acid and other dicarboxylic acid aminoacids, and other tri- or poly-functional amino acid derivatives.

In accordance with another embodiment of the invention, a composition ofthe present invention may be administered alone, or in combination(sequentially or in batch) with other immunotherapeutic compositions.The sonosensitizers of the present invention may be further combinedwith agents for other activation modalities, e.g., photosensitizersand/or radiation sensitive agents. These features afford potentialaugmentation of the sonodynamic therapeutic ratio through sequentialsensitizer administration (followed by sound activation). Under theseconditions, a distant metastasis may be targeted.

In this embodiment of the invention, a SDT method comprisesadministering a first sonodynamic agent, preferably having a slowuptake, and administering a second sonodynamic agent, preferably havinga more rapid uptake than that of the first agent. Both first and secondsonodynamic agents may then be activated by exposing the patient and/orthe agent to sound of suitable frequency, as described above.

The excellent fluorescence properties of the hypocrellins andderivatives provide a valuable tool to monitor intracellular uptake anddistribution kinetics by confocal laser scanning microscopy (CLSM). Eachdrug has unique properties of uptake and distribution (Miller et al 1995a,b). The rate cells take up drug in humans in vitro and in vivo can bedetermined using similar protocols as Liu et al 1995 and Miller et al.,1995 a or b).

Some of the embodiments of the present invention also have the addedbenefit of functioning with or without the presence of oxygen.Therefore, some embodiments of the present invention are effective inthe treatment of solid tumors which are either well oxygenated or eitherpartially or fully hypoxic.

The sono-activating agents may be formulated for topical application inpenetrating solvents or in the form of a lotion, cream, ointment or gelcontaining a sufficient amount of the sonosensitizing agent compound tobe effective for SDT therapy. Such topical formulations may be preparedin gel form by combining the photosensitizing agent with a solvent andadding a gelling agent thereto. Suitable gelling agents includecarboxymethyl cellulose (Carbopol.TM. 934P from B. F. Goodrich ofBrecksville, Ohio U.S.A.) and fumed silica (CAB-O-SIL.RTM., Cabot Corp.,Tuscola, Ill.). The gelling agent is generally used in amounts of about5-10 wt % to obtain a gel with the desired viscosity. Obviously, gelscontaining more or less gelling agent will have slightly higher or lowerviscosity. One skilled in the art can readily obtain the desired gelviscosity by adjusting the concentration of gelling agent.

Additives, such as cosolvents, surfactants and/or bioadhesivesfrequently improve the gel properties and may be added as desired.Suitable cosolvents/surfactants include propylene glycol and glycerine.Suitable bioadhesives include carboxymethylcellulose, polyacrylicpolymers, chitosan and sodium alginate, modified starch with polyacrylicpolymers, eudispert hv hydrogels or xerogels, sodium hyaluronate, andpolymers of polyethylene glycol, hydroxypropylcellulose, orcarboxyvinyl. The additives may be incorporated into the gel bymechanically mixing the additives into a mixture of solvent and gellingagent.

Other additives may be used to enhance or maintain chemical stabilityand physiological suitability. Examples are antioxidants, chelatingagents, inert gases, buffers and isotonicifiers. Examples ofantioxidants and typical concentration ranges include acetone sodiumbisulfite (0.1-0.8%), ascorbic acid (0.05-1.0%), monothioglycerol(0.1-1.0%), potassium metabisulfite (0.05-0.1%), propyl gallate (0.02%),sodium bisulfite (0.01-1.0%), sodium formaldehyde sulfoxylate(0.03-0.1%), sodium metabisulfite (0.02-0.25%), sodium sulfite(0.01-0.1%), sodium thioglycolate (0.05-0.1%).

Examples of chelating/complexing agents and typical concentration rangesinclude edetate sodium (0.005-0.1%), edetate calcium disodium(0.005%-0.01%), gentisic acid ethanolamide (1.0%-2.0%), niacinamide(1.0%-2.5%), sodium citrate (0.01%-2.5%), citric acid (0.001%-1.0%).

Buffers are used primarily to stabilize a formulation against thechemical degradation that might occur if the pH changed appreciably.Buffer systems employed normally have as low a buffer capacity asfeasible in order to not disturb significantly the body buffer systemswhen injected. The buffer range and effect of the buffer on activitymust be evaluated. Appropriate adjustment is useful to provide theoptimum conditions for pH dependent partition into the target malignanttissues or lesion area. Examples of such buffer systems include thefollowing acids: acetic, adipic, ascorbic, benzoic, citric, glycine,lactic, tartaric, hydrochloric, phosphoric, sulfuric, carbonic andbicarbonic; and their corresponding salts such as: potassium, sodium,magnesium, calcium and diethanolamine salts.

When the solution will be dispensed from multiple dose containers,antimicrobial agents in bacteriostatic or fungistatic concentrations areadded in amounts effective to provide protection from bacteria or fungi.Among the compounds and concentrations most frequently employed arephenylmercuric acid (0.002-0.01%), thimerosal (0.01%), benzethoniumchloride (0.01%), benzalkonium chloride (0.01%), phenol or cresol(0.5%), chlorbutanol (0.5%), benzyl alcohol (2.0%), methylp-hydroxybenzoate (0.18%), propyl, p-hydroxybenzoate (0.02%), andethylenediaminetetraacetic acid (EDTA).

Suitable penetrating solvents are solvents for the porphycene compoundwhich will enhance percutaneous penetration of the porphycene compound.Solvents which have this property include proparacaine, dimethylsulfoxide, dimethyl acetamide, dimethylformamide,1-methyl-2-pyrrolidone, diisopropyladipate, diethyltoluamide and to alesser extent propylene glycol. Additional solvents include substitutedazacycloalkan-2-ones having from 5 to 7 carbons in the cycloalkyl groupsuch as 1-dodecylazacycloheptan-2-one (AZONE) and otherazacycloalkan-2-ones such as described in U.S. Pat. No. 3,989,816incorporated herein by reference. Also included areN-bis-azocyclopentan-2-onyl alkanes described in U.S. Pat. No. 3,989,815(hereby incorporated by reference), 1-substituted azacyclopentan-2-onesdescribed in U.S. Pat. No. 3,991,203 (hereby incorporated by reference)and water-soluble tertiary amine oxides described in U.S. Pat. No.4,411,893 (hereby incorporated by reference).

The sonosensitizing agents can be used with solvents and adjuvantsappropriate to the sonosensitizing agent chemistry to adjust theviscosity of the formulation. The most important solvents in this groupare ethanol, polyethylene glycols of the liquid series and propyleneglycol. A more comprehensive listing includes acetone, dimethylacetamide, dimethyl formamide, dimethyl sulfoxide ethanol, glycerin,polyethylene glycol 300, and 400, propylene glycol, sorbitol,polyoxyethylene sorbitan fatty acid esters such as laureate, palmitate,stearate, and oleate, polyoxyethylated vegetable oil, sorbitanmonopalmitate, 2-pyrrolidone; n-methyl-2-pyrrolidine;n-ethyl-1-pyrrolidine; tetrahydrofurfuryl alcohol, tween 80 and dimethylisosorbide. Dimethyl isosorbide (ARLASOLVE®. DMI, ICI SpecialtyChemicals) has the advantage of being both water- and oil-soluble.Additionally, dimethyl isosorbide may be readily gelled with a gellingagent to produce gel formulations with, for example, 4% KLUCEL.®(Hercules).

Additional topical formulations which may be used for the chosensonosensitizing agent are disclosed in U.S. Pat. Nos. 3,592,930 and4,017,615 which are hereby incorporated by reference.

EXAMPLES Example 1 Screening Assay for Efficiency of Sonosensitizers inVitro

This screening assay makes use of human promyelocytic leukemia cellsgrowing in suspension culture. The transducer of an UltraMax ultrasoundgenerator for physiotherapy was adapted for in vitro studies by fittinga tissue culture-compatible Teflon cuff such that cell suspensions couldbe conveniently insonated. The following diagrams represent theequipment employed.

Human acute promyelocytic leukemia HL-60 cells (ATCC No CCL-240) weremaintained in RPMI 1640 cell culture medium supplemented with 7 g/L ofL-glutamine (Gibco, Grand Island N.Y.), 10% fetal bovine serum(Cansera), and 1% penicillin-streptomycin-neomycin (Gibco), bufferedwith NaHCO₃ to pH 7.4. They were incubated at 37° C. in a humidifiedatmosphere of 95% air, 5% CO₂, and maintained at cell densities between2.0×10⁵ to 1.0×10⁶ cells/mL. For insonations in the presence of Hp (aporphyrin positive control) or hypocrellin test compounds, cells werebrought to approximately 1.0×10⁶ cells/mL, incubated with the putativesensitizer at 37° C. for 2 hours in T-flasks, equilibrated for 30minutes at room temperature, resuspended and insonated. All procedurestook place in low-light conditions to avoid spurious photosensitization.Cells were gently resuspended for 10 seconds before insonation. The testsonosensitizer was dissolved in dimethylsulfoxide (DMSO) such that thefinal concentration of DMSO in the insonation medium did not exceed 1%(v/v). Stock sensitizer was prepared by slowly adding RPMI-1640 mediumto the appropriate volume at final sensitizer concentrations between 0.3mM and 1 mM.

Sonodynamic Therapy Survival Assay:

Aliquots of cells were withdrawn prior to insonation to serve as theoriginal value for cell survival. Cells were suspended in Isoton diluentand counted in a Coulter electronic particle counter. Insonationproceeded at a power density of 2 W, at times varying between 3- and 10seconds to effect cytotoxicity by sonosensitization. Control cultureswere subjected to the appropriate dose of ultrasound, in the presence ofDMSO, and the absence of the test sonosensitizer. Following ultrasoundtreatment, aliquots of the cell suspension were withdrawn for countingas above, and the survival fraction, S, was calculated as the fraction:S=(Cell number following insonation/cell number prior toinsonation)×100.Results:

We are currently interested in demethoxy hypocrellin B (DMHB) as asonosensitizer, based on its excellent properties of photosensitization.Standard dose-response curves, with a fixed dose and energy ofultrasound demonstrate proof-of-concept that DMHB is an extremelyefficient sonosensitizer of human cells in vitro. The LD₅₀ of thepositive hematoporphyrin (Hp) control is 1.0 mM, whereas the LD₅₀ forDMHB is approximately 100 μM, a 10-fold difference.

Conclusion:

DMHB has high potential as an effective sonosensitizer in vivo. DMSODMSO + DMSO + DMSO + Only 1 μM DMHB 10 μM DMHB 100 μM DMHB Survival,55.8 41.8 37.3 30.7 % 53.0 48.2 43.3 39.4 Mean 40.0 33.5 24.9 23.7 49.641.1 35.2 31.3 s.d. 8.4 7.3 9.4 7.9 Mean - 100.0 83.0 71.1 63.2 DMSO Bg

Example 2 Efficiency of DMHB in Vivo

We explored the use of DMHB in Balb/c mice engrafted in the flank withthe syngeneic murine mastocytoma, EMT6. The mice were inoculatedsubcutaneously with 1.0×10⁵ EMT6 tumor cells in sterile injectablesaline. Within 7 days, detectable tumors are evident. Mice wererandomized into a control group and a SDT treatment group, whichdiffered only in that the sonosensitizer was not present in the drugsolvent (see below). Fresh DMHB was dissolved in mineral oil, andinjected intraperitoneally into each mouse to achieve a total bodyconcentration of 50 μM. Following a an interval of 48 h forbiodistribution of the sensitizer, the mice were lightly anesthetizedwith Metafane, and placed on a 2% agarose gel, with the tumor lying in adepression of the gel which was juxtaposed to the transducer of anUltraMax physiotherapy ultrasound generator. Ultrasound was applied at afrequency of 1 MHz, at a power density of 0.2 W/cm², for a 10-minuteperiod. The maximum energy applied to the tumor volume was approximately500 J. Tumor growth was monitored daily by caliper measurements in threemutually orthogonal planes. Tumor volume was calculated from theexpression,V=π/6×d₁×d₂×d₃where V=volume (mm³), and d_(1,2,) and ₃ are the three mutuallyorthogonal diameters. The endpoint for the assay was the proportion oftumors reaching 4 times treatment volume, which was plotted as afunction of time. Animals were euthanized at this point. FIG. 2 is aplot of tumor growth vs. time.

Example 3

HL-60 cells were treated with perylenequinone sensitizers and insonatedas described above. The surviving fractions were plotted againstsensitizer concentration. At a concentration of approximately 30 μM,CPMg(Ac₂) showed sonotoxicity exceeding that of the 1000 μM Hp control,with the decrease in survival occurring steeply over the preceding twodecades of sensitizer concentration. DBHB and DMHB showed negligiblesonotoxicity up to 100 μM; the bulk of the observed sonotoxic effectoccurred over the decade from 100 μM to 100 μM, and the maximum effectwas comparable to that of the Hp control. HBMg(Ac₂) showed no sonotoxiceffect until 10 μM. Cell survival decreased steeply over the next twodecades of sensitizer concentration.

Example 4 Perylenequinonoid Pigments (Hypocrellins) and TheirPhotosensitizing and Sonosensitizing Properties

Photo- sensi- tizing Sonosensitizing Compound Potential* Potential*DMHBa Demethylated-HB 3.0 μM 1.0 mM DMHBb 2-butylamino-2-demethoxy- 0.1μM 0.1 mM Hypocrellin B HA Hypocrellin A 4.0 μM None HBAC-R1Cystamine-HB isomer 1 1.0 μM None HBAC-R2 Cystamine-HB isomer 1 5.0 μMNone HBAM-R1 2-morpholino-ethylaminated 4.0 μM None HB HBDD-R12-(N,N-dimethyl-amino) 1.0 mM propylamine-Hypocrellin B HBEA-R1Ethanolamine-Hypocrellin B 0.15 μM  1.0 mM isomer 1 HBEA-R2Ethanolamine- 7.50 μM  None Hypocrellin B isomer 2 HBED-R2Ethylenediamne- 4.0 μM None Hypocrellin B HBMA-IV Methylamine- 1.0 μMNone Hypocrellin B*Molar Concentration which exerts LD₅₀ in EMT6 murine mastocytoma invitro, for a fixed dose of light or ultrasound

Example 5

150 mg Hypocrellin B (0.28 mmol) is dissolved in 100 mL fresh distilledbenzene containing excessive cyclopentylamine and the resulting solutionis stirred for 18 hours. The solvent is removed under reduced pressure.Chloroform is used to wash the residue for 3-4 times, and the chloroformlayer is washed with water for 3-4 times. Chloroform is evaporated, thenthin-layer chromatography (TLC) is used to purify the residue using3:1:0.5 (V:V:V) cyclohexane-ethyl acetate-95% ethanol as eluent. TLC isrepeated twice for further purification, the amino-substituteddemethoxylated hypocrellin B is obtained.

Characteristic of the product:

UV-vis spectra (λ_(max)): 466 nm, 577 nm, 641 nm;

IR spectra (v_(max)): 3419 cm⁻¹, 2922 cm⁻¹, 1680 cm⁻¹, 1608 cm⁻¹;

1 HNMR (δ): 6.32 (s), 6.80 (s), 3.08 (s), 2.55 (s), 1.79 (s), 1.91 (s),4.01 (s), 4.0 (s), 4.24 (s), 1.86 (m), 1.68 (m), 1.49 (m), 1.00 (m),16.07, 15.78 ppm;

Mass spectra (m/z): 583 (M⁺)

Example 6

Hypocrellin B (HB) was prepared by quantitative potassium hydroxidedehydration of hypocrellin A (HA) followed by neutralization with Ha,chloroform extraction, and recrystallization with benzene-petroleumether, 2-butylamino-2-demethoxy-hypocrellin B (2-BA-2-DMHB) was preparedby reflux with n-butylamine in pyridine, neutralization, and chloroformextraction of HB. The product was subjected to 1% citric acid-silica gelthin-layer chromatography (TLC), using a 6:1:1 mixture of petroleumether/ethyl acetate/ethanol (95%) as eluent, and three compounds wereobtained. They were the target compound (rate of flow (Rr)=0.64) and twoby-products (Rr=0.74 and 0:40. respectively), which were identified bysatisfactory NMR, mass spectra and elemental analysis [12]. The targetcompound was further purified with TLC and the desired product,2-BA-2-DMHB, was obtained in 54% yield. The purity or HB and 2-BA-2-DMHBwas assessed by high-performance liquid chromatography and found to behigher than 95%. The chemical structure of 2-BA-2-DMHB is shown inFIG. 1. The sonosensitizers were kept in lyophilized form until the dayof experiments, at which time they were dissolved in DMSO.

While the invention has been described in some detail by way ofillustration and example, it should be understood that the invention issusceptible to various modifications and alternative forms, and is notrestricted to the specific embodiments set forth. It should beunderstood that these specific embodiments are not intended to limit theinvention but, on the contrary, the intention is to cover allmodifications, equivalents, and alternatives falling within the spiritand scope of the invention.

1. A method of treatment comprising administering a compositioncontaining an amino-substituted hypocrellin compound, and activating thecompound by exposing the compound to sound of a pre-determinedfrequency.
 2. The method of claim 1 wherein the amino-substitutedhypocrellin compound comprises an effective amount of a demethoxylatedhypocrellin B.
 3. The method of claim 2 wherein the demethoxylatedhypocrellin B is 2-butylamino-2-demethoxy-hypocrellin B.
 4. The methodof claim 1 wherein the method of treatment comprises treating skinconditions, cancer, viral diseases, retroviral diseases, bacterialdiseases, autoimuune diseases and fungal diseases.
 5. The method ofclaim 1 wherein activating the composition comprises exposing thederivative to sound.
 6. The method of claim 5 wherein exposing thederivative to sound comprises exposing the derivative to a frequencybetween about 50 kHz and about 12 MHz.
 7. The method of claim 6 whereinexposing the derivative to sound comprises exposing the derivative to afrequency between about 1 MHz and about 3 MHz. Page 3
 8. The method ofclaim 1 further comprising activating the compound by exposing thecompound to light of a pre-determined wavelength.
 9. A method fortreating a disease or condition comprising administering a compositioncomprising a sonosensitizer hypocrellin derivative and at least one of apKa modifier, a buffer, a salt, a base, an acid, saline, and an adjuvantand activating said composition by exposing the composition to sound ofa pre-determined frequency.
 10. A therapeutic composition containing asonosensitizer comprising a demethoxylated hypocrellin, wherein thedemethoxylated hypocrellin is 2-butylamino-2-demethoxy-hypocrellin B.11. (canceled)
 12. The method of claim 9 wherein the sonosensitizerhypocrellin derivative is a demethoxylated hypocrellin.